preLighters’ choice – October’s handpicked preprints

We’ve launched a new preLights initiative: each month, preLighters with expertise across developmental and stem cell biology nominate a few recent developmental and stem cell biology (and related) preprints they’re excited about and explain in a single paragraph why. Short, snappy picks from working scientists — a quick way to spot fresh ideas, bold methods and papers worth reading in full. These preprints can all be found in the October preprint list.

October highlights

• Deevitha Balasubramanian
• Dillan Saunders
• Jawdat Sandakly
• Mansi Srivastava
• Manuel Lessi
• Sristilekha Nath
• Theodora M Stougiannou

Deevitha Balasubramanian

Preprint:

Multiplexed embryo profiling links cellular state to zygotic genome activation in single cells
Max Hess, Marvin F. Wyss, Edlyn Wu, Joel Lüthi, Chiara Rebagliati, Nadine L. Vastenhouw, Darren Gilmour, Shayan Shamipour, Lucas Pelkmans

preLight:

Have you ever wanted to image dozens of your favorite proteins, together, in 3D, and at single-cell resolution?

This preprint describes the development of a 3D adaptation of the previously described iterative indirect immunofluorescence imaging (4i) technique and its application to early zebrafish embryos to explore the heterogeneity in the onset of zygotic genome activation (ZGA). 3D-4i enables multiplexed immunofluorescence and in toto imaging of whole-mount structures, allowing high-resolution and high-throughput visualization of proteins and is supported by a comprehensive image analysis pipeline. Using 3D-4i, the authors capture the levels of proteins like cell cycle regulators, histone modifications, pluripotency factors, and RNA polymerase II at single-cell resolution. This leads to many key findings, including a framework to infer cell cycle phase and accurately predict transcriptional output, revealing how multiple features act collectively to precisely modulate ZGA onset.

Dillan Saunders

Preprint:

A toolkit for testing membrane-localising tags across species
Irene Karapidaki, Mette Handberg-Thorsager, Tsuyoshi Momose, Hitoyoshi Yasuo, Grigory Genikhovich, Sarah Assaf, Clara Deleau, Ying Pang, Clayton Pavlich, Beke Lohmann, Maria Lorenza Rusciano, Mattia Stranges, Juliette Mathieu, Marie Zilliox, Kirill Ustyantsev, Bastien Salmon, Béryl Laplace-Builhé, Manon Koenig, Jeffrey J. Colgren, Maria Ina Arnone, Eugene Berezikov, Thibaut Brunet, Gregor Bucher, Pawel Burkhardt, Daniel J. Dickinson, Evelyn Houliston, Jan Huisken, Lucas Leclère, Michalis Averof

preLight:

All things bright and beautiful. 

Advances in technology have made detailed study of non-model organisms more feasible, yet there is always a challenge in applying existing techniques to new systems. The authors take a systemic approach to screening a varied set of membrane-localisation tags in the early embryos of a wide range of organisms. They identify several tags that display strong, membrane specific fluorophore localization in many species but highlight that no single tag is ubiquitously successful. This work is an open science project that combines the efforts of many labs to provide a useful community resource. Check this preprint out if you’re looking to label membranes in your species of interest!

Jawdat Sandakly

Preprint:

Post-translational Tuning of Human Cortical Progenitor Neuronal Output
Julien Pigeon, Tamina Dietl, Myriame Abou Mrad, Ludovico Rizzuti, Miguel V. Silva, Natasha Danda, Corentine Marie, Clarisse Brunet Avalos, Hayat Mokrani, Laila El Khattabi, Alexandre D. Baffet, Diogo S. Castro, Carlos Parras, Boyan Bonev, Bassem A. Hassan

preLight:

A novel role for PTMs in fine tuning neurogenesis.

While human brain development has long been linked to alterations in genomic sequence, the authors raise the question: do post translational modifications (PTMs) offer a complementary mechanism in shaping human brain evolution ? In particular,  they focus on Neurogenin 2 (NEUROG2), a master regulator of neural fate and neuronal identity specification, whose activity is dependent on PTMs such as phosphorylation. Through a combination of genome editing, high-throughput imaging, and single-cell multiomics, they investigate whether NEUROG2 has evolved species-specific functional plasticity in human radial glial cells (RGCs). They find that the human NEUROG2 regulates both deep and upper layer neuron production and controls the balance between proliferative and neurogenic divisions in RGCs via its phosphorylation at residue T149. This phosphorylation tunes AP-1 (JUN/FOS) driven gene regulatory networks in RGCs, enhancing neurogenic commitment and increasing upper-layer neuron production. Phospho-mutant NEUROG2 promotes premature chromatin opening at AP-1 binding sites, priming RGCs for differentiation without accelerating neuron maturation. Overall, their findings suggest that the evolutionary innovations in brain development do not solely rely on genetic changes but can also arise through modifications of conserved proteins.

Mansi Srivastava

Preprints:

A conserved logic for the development of cortical layering in tetrapods
Astrid Deryckere, Saket Choudhary, Connor Lynch, Lian Kirit V. P. Limperis, Pauline Affatato, Jamie Woych, Elias Gumnit, Alonso Ortega Gurrola, Rahul Satija, Christian Mayer, Maria Antonietta Tosches

&

An atlas of shark developing telencephalon reveals ancient origin of basal progenitors and Cajal-Retzius cells
Idoia Quintana-Urzainqui, Tobias Gerber, Phillip A. Oel, Leslie Pan, Nikolaos Papadopoulos, Z. Gülce Serka, Ana Verbanac, Maite Börsig, Dorinda Torres-Sabino, Isabel Clara Rollán-Delgado, Luca Santangeli, Henrik Kaessmann, Detlev Arendt

preLight:

Shark and Salamander – pioneers in building beautiful brains.

“Evolutionary change is often driven by changes in development.”

Most of our understanding of brain development comes from studying the mouse (mammal). However, a tubular brain and backbone are the defining features of a subphylum – vertebrata. What if mammalian brain development actually combines elements found in the brain developmental programs of fish, salamanders, and birds, much like a musical genre blending influences from several styles?
These two papers dig into shark and salamander brains using a combination of single-cell RNA sequencing, spatial transcriptomics, birth-dating, lineage tracing, and computational approaches to learn the origin of brain development as we know it.

Combining these two papers is as satisfying as putting together a jigsaw puzzle.

• The shark brain has field-level homology with that of the salamander and mouse.
• Both sharks and salamanders have multipotent progenitors that give rise to intermediate progenitors: the driving force behind big brains.
• The Cajal-Retzius cells marked their enigmatic presence in sharks.
• In salamander, the molecular identity, layer position, and projection are functions of birthdate.

With this prologue, dive into the many observations these two papers make, and discuss where the point of difference arose in the vertebrates that made their brains look and behave differently. 

Manuel Lessi

Preprint:

IPSC-based modeling of resiliency in centenarians reveals longevity-specific signatures
Todd W. Dowrey, Samuel F. Cranston, Nicholas Skvir, Yvonne Lok, Payton Bock, Elizabeth K. Kharitonova, Elise MacDonald, Ella Zeldich, Christopher Gabel, Alexander Tyshkovskiy, Stefano Monti, Vadim N. Gladyshev, Paola Sebastiani, Thomas T. Perls, Stacy L. Andersen, George J. Murphy

preLight:

Giving new life to elderly cells reveals what makes them resilient to aging.

Understanding the molecular and cellular mechanisms that govern aging has been, and will likely remain, a central question for humanity. What better way to approach this challenge than by studying individuals who appear to defy canonical aging mechanisms? In this study, the authors generated pluripotent stem cells from a cohort of centenarians and differentiated them into excitatory cortical neurons. Molecular and cellular comparisons with neurons from non-centenarian individuals revealed that centenarian-derived neurons exhibit a distinct resilience signature, marked by enhanced synaptic integrity, calcium homeostasis, and energy-efficient metabolism at baseline. When challenged, these neurons demonstrated superior dynamic stress responses, in contrast to non-centenarian neurons, which showed chronic proteostatic stress activation and blunted responsiveness. Overall, this work highlights the versatility of the stem cell platform in uncovering molecular mechanisms that confer resilience to aging in neural systems. This represents a foundational resource for investigating the determinants of aging across diverse cell types and developmental contexts, leveraging the innate ability of stem cells to recapitulate key human developmental processes.

Sristilekha Nath

Preprint:

Tissue surface mechanics constrains proliferation-driven forces to guide mammalian body axis elongation
Marc Trani-Bustos, Ryan G Savill, Arthur Boutillon, Petr Pospíšil, Deniz Conkar, Claudia Froeb, Johannes R Soltwedel, Heidi L van de Wouw, Ellen M Sletten, Jesse V Veenvliet, Otger Campàs

preLight:

Decoding mammalian body axis elongation: a supracellular ‘actin cap’ in action.

Body axis elongation is fundamental to establishing a head-to-tail body plan in vertebrates, including mammals. Although the genetic and biochemical pathways involved are well studied, the physical forces that help shape the mammalian axis remain less understood. To investigate these mechanisms, the authors of this preprint used mouse and human stem-cell-derived gastruloids, an accessible model that bypasses the challenges of working with embryos in utero. By integrating a previously developed gastruloid analysis framework and oil droplet-based deformation measurements, the authors aimed to uncover the mechanical forces at play. Their findings show that randomly oriented cell divisions generate isotropic expansive forces throughout the gastruloid during the elongation period. However, a posteriorly enriched actin network, termed the ‘actin cap’, provides localized mechanical resistance, preventing tissues at the posterior domain from expanding laterally, thereby guiding the elongation of the body axis.  Apart from mouse and human gastruloids, mouse embryo explants display similar proliferation and actin patterns, supporting the idea that this actin cap–based mechanical constraint is a conserved and previously overlooked mechanism in mammalian axis elongation. 

Theodora M Stougiannou

Preprint #1:

Generation of parasympathetic neurons from hiPSC that reproduce the electrophysiological properties of native neurons and modulate the activity of hiPSC-atrial cardiomyocytes
Alison M. Thomas, Isabella Noelle Chiong, Joana F Neves, Andrew Tinker, Franziska Denk,  Laura Fedele

preLight:

What if we could use patient cells to generate neurons that can replace dysfunctional native cells and tackle diseases characterised by the aberration of atrial electromechanical activity, namely atrial fibrillation? This is the question the authors of this manuscript in preprint answer, a detailed protocol for the derivation of parasympathetic neurons from human induced pluripotent stem cells (hiPSC). The described protocol includes several useful features, including the lack of batch-testing for growth factors as well as the integration of electrophysiological and functional assessment testing to specifically identify the presence of parasympathetic neurons. The authors also describe cellular features the user should look out for, to ensure proper progression through the protocol steps, including the presence of smooth spheroids during the Embryoid bodies stage and the presence of neuronal-like projections during the neuronal differentiation stage, the expression of specified markers of autonomic (ASCL1, PHOX2B) and parasympathetic (CHAT, VACHT) populations and markers of autonomic neuron development (ISL1). The protocol also includes troubleshooting sections, which is sure to help new users make the most of it.

Preprint #2:

ETV2 mediated differentiation of human pluripotent stem cells results in functional endothelial cells for engineering advanced vascularized microphysiological models
Shun Zhang, Zhengpeng Wan, Lei Wang, Caihong Wu, Junkai Zhang, Sarah Spitz, Xun Wang, Marie A. Floryan, Mark F Coughlin, Francesca M. Pramotton, Liling Xu, Ron Weiss, Roger D. Kamm

preLight:

Water must flow, but will blood in in vitro models do the same? In this study, the authors generate endothelial cells (EC) derived from human induced pluripotent stem cells (hiPSC); efficiency of hiPSC-derived EC generation is enhanced via overexpression of ETV2, a factor involved in vascular and cardiac development. In short, expression of ETV2 is induced in the hiPSC lines used; these are then subjected to differentiation protocols that will eventually generate EC. In vitro, these same cell lines can self-assemble into stable and lumenized microvascular networks (MVN) on the surface of microfludic chips; more importantly, however, no such success in vascular formation has been observed in lines subjected to the conventional differentiation models, highlighting the importance of growth factor overexpression in pluripotent source populations. This study provides an answer to the problem of organoid vascularization and can be applied in models examining tumor vascularization as well as models evaluating the blood brain barrier (BBB).

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